Metabolic Syndrome and Combination Antiretroviral Therapy in HIV Patients in Periurban Hospital in Ghana: A Case-Control Study

Background There is an increasing prevalence of cardiovascular diseases (CVDs) and risk factors in HIV patients as the levels of AIDS-related mortality and morbidity decrease. Metabolic syndrome (MetS) is the accumulation of various CVD risk factors that predict the occurrence of CVDs. We investigated the prevalence of MetS and associated risk factors in HIV patients treated with combination antiretroviral therapy (cART), cART-naïve HIV patients, and non-HIV controls. Methods In a case-control design, 158 cART-treated HIV patients, 150 cART-naïve HIV patients, and 156 non-HIV controls were recruited from a periurban hospital in Ghana. A structured questionnaire was used to collect data on demography, lifestyle, and medication. Anthropometric indices and blood pressure were measured. Fasting blood samples were collected to measure the plasma levels of glucose, lipid profile, and CD4+ cells. The presence of MetS was defined using the joint scientific statement criteria. Results The prevalence of MetS was higher in cART-treated HIV patients compared with cART-naïve HIV patients and non-HIV controls (57.3% vs. 23.6% vs. 19.2% and p < 0.001, respectively). MetS was associated with cART-treated HIV patients (odds ratio (95% CI) = 7.24 (3.41–15.39) and p < 0.001), cART-naïve HIV patients (2.04 (1.01–4.15), p=0.048), and female gender (2.42 (1.39–4.23) and p=0.002). In cART-treated HIV patients, those on zidovudine (AZT)-based regimens were associated with increased likelihood (3.95 (1.49–10.43) and p < 0.006), while those on tenofovir (TDF)-based had decreased likelihood (0.32 (0.13–0.8) and p=0.015) of having MetS. Conclusion In our study population, there was a high prevalence of MetS in cART-treated HIV patients compared to cART-naïve HIV patients and non-HIV controls. HIV patients on AZT-based regimens had an increased likelihood of having MetS, while those on TDF-based regimens had a reduced likelihood of having MetS.


Introduction
Sub-Saharan Africa (SSA) has the highest population of people living with HIV (PLWH) worldwide and this is associated with healthcare, socioeconomic, and developmental challenges [1]. It was reported in 2017 that 71% of the global total number of PLWH resides in SSA, with 75% of HIVrelated deaths and 65% of new HIV infections occurring in this region [2]. In Ghana, the prevalence of HIV infection is 1.7% and more concentrated in urban and periurban areas [3]. Te widespread availability and accessibility of combination antiretroviral therapy (cART) in low-middleincome countries have dramatically decreased the mortality attributable to HIV infection in the past decade [4]. Now, PLWHs live longer and become more susceptible to chronic diseases, especially cardiovascular diseases (CVDs) [5,6].
Te development of CVDs in patients and the general population has been associated with metabolic syndrome (MetS), which is defned as the accumulation of some cardiometabolic risk factors such as abdominal obesity, high blood pressure, dyslipidemia, and impaired fasting plasma glucose [7,8]. Te prevalence of MetS has been reported to be higher in PLWH compared to the general population [9,10]. Even in PLWH, there is substantial variation in the prevalence of MetS, mainly due to diferent diagnostic criteria for MetS used across various studies, as well as diferences in the sociocultural attributes of the population studied [11]. A global meta-analysis of MetS, in which only two studies from Africa were included, reported the estimated prevalence of MetS to be 29.6% [12]. A recent metaanalysis of the Sub-Saharan African population reported an estimated prevalence of MetS in PLWH to be 21.5% with substantial heterogeneity across various studies analysed [6]. In Ghana, there has been only one study that has reported the prevalence of MetS in the Ghanaian PLWH population [13].
Te current treatment guidelines for HIV in Ghana require that all PLWH be treated with combination antiretroviral therapy (cART) irrespective of their disease staging and/or CD4+ lymphocyte count. Te recommended frst line treatment is tenofovir (TDF)-based regimen, combined with either lamivudine (3TC)/emtricitabine (FTC) or efavirenz (EFV)/nevirapine (NVP). However, a substitute for the TDF-based regime, zidovudine (ZDV)based regimen, is widely used in managing PLWH in Ghana, especially during the past 5 years, when there was a shortage of TDF-based regimen. Protease inhibitor-based regimens are not commonly used among most PLWHs in Ghana [14]. It has been reported that these cART regimens are associated with MetS and CVDs, but these study reports are from patients in the developed countries [11,15] and the Asian population [5]. Tere is no study reporting the association between cART regimens and MetS in the Ghanaian population. In this study, we compared the prevalence and associated factors of MetS in cART-treated and cART-naïve HIV patients with non-HIV participants. We also assessed the association between various cART regimens and MetS in Ghanaian PLWH.

Study Participants, Site, and Design.
Tis study was a case-control design with HIV patients as cases and the controls were non-HIV individuals who visited the HIV clinic for voluntary testing of their HIV status. HIV patients were recruited by a systemic random sampling as every third consenting patient was invited to join the study, whereas non-HIV participants were recruited conveniently by invitation. HIV patients were categorized as those on cART management (cART-treated) and newly diagnosed patients who were yet to be put on cART medication (cART-naïve). Te study was conducted at Atua Government Hospital, a 150-bed primary healthcare facility, located in Agormanya, a periurban town in the Eastern region of Ghana. Te Agormanya area has a high prevalence of HIV infection compared to the national prevalence, and the hospital has about 1,400 HIV patients on its register. Ethical approval was obtained from the College of Health Sciences Ethical and Protocol Review Committee (CHS-Et/M.6-5.17/2018-2019), and all participants provided voluntary informed consent before joining the study.

Data Collection.
A structured questionnaire was used to obtain data on sociodemographic factors such as age, gender, lifestyle factors (smoking and alcohol intake), medical history (hypertension, diabetes, and cardiovascular disease), current cART medication, occupation, education (school cycle completion), and marital status. Smoking status was classifed as never, past (smoking cessation since more than 1 year before the survey), or current smoking, and alcohol intake was classifed as drinkers and nondrinkers. Te body weight and height were measured using a stadiometer in light clothing with footwear removed. Te waist and hip circumferences were measured with a nonelastic tape measure parallel to the foor without compressing the skin. Pulse rate and systolic and diastolic blood pressure (average of two measures for each arm at 1-min intervals) were recorded using a semiautomatic blood pressure monitor (BF-508, Omron Healthcare, Inc., Vernon Hills, IL, USA). Hypertension was defned in the case of self-reported ongoing antihypertensive treatment and/or systolic blood pressure ≥140 mm Hg and/or diastolic blood pressure ≥90 mm Hg.

Biochemical Analysis.
After 8-12 h of overnight fasting, approximately 5 ml of venous blood was drawn from the antecubital fossa into appropriate tubes. Te samples were centrifuged at 4000 G, and the serum/plasma was aliquoted and stored at −70°C until analysis. Fasting plasma glucose (FPG), total cholesterol, high density (HDL) lipoprotein cholesterol, and plasma triglyceride levels were analysed using a biochemistry analyser (Contec BC 400, China) and commercial reagents (Randox Laboratory Reagents, UK). Low-density lipoprotein (LDL) cholesterol levels were calculated using Friedewald's formula. Te CD4 cell count was measured using TriTEST reagents following an in-house, dual platform protocol, and multiset and attractors software using a FACScan fow cytometer (Becton-Dickinson, NJ, USA).

Defnition of MetS.
Te defnitions of MetS based on the criteria of the Joint Interim Statement (JIS), the National Cholesterol Education Program Adult Treatment Panel III (ATP III) [16], and the International Diabetes Federation (IDF) [17] were initially used. IDF defnition of MetS is based on waist circumference (adjusted for Africans) >80 cm in women and >94 cm in men plus two of the following: triglycerides ≥1.7 mmol/l or specifc treatment for this lipid abnormality, HDL <1 mmol/l in males and <1.3 mmol/l in females, FPG >5.6 mmol/l or previously diagnosed type 2 diabetes, systolic blood pressure ≥130 mmHg, or diastolic blood pressure ≥85 mmHg. ATP III defnition is based having three or more of the following: waist circumference >88 cm in women and >102 cm in men, triglycerides ≥1.7 mmol/l, HDL <1.3 mmol/l in women or <1 mmol/l in men, FPG >6 mmol/l, or blood pressure ≥130/≥85 mmHg.
JIS defnition attempted to reconcile the ATP III and IDF criteria, with the criteria being similar to IDF, except that elevated waist circumference is no longer a mandatory requirement [18]. Tis study adopted the JIS criterion in analysis of MetS.

Sample
Size. Te minimum sample size required was computed with the online Epitools for a case-control study design. We assumed that the prevalence of MetS among non-HIV controls would be 20% and the HIV patients would have an odds ratio of 2.2 for MetS [8,11,13]. At the 95% signifcant level and 80% power, at least 128 participants were required for each group. We, therefore, targeted to recruit a minimum of 150 participants in each group for the study.

Data Analysis.
Te data were analysed using SPSS version 27. Te comparisons of anthropometric indices, biochemical analytes, sociodemographic, and clinical variables were performed using ANOVA for continuous variables with normal distribution and the Kruskal-Wallis H test for variables with non-normal distribution. Association between categories of study participants versus MetS and its components were analysed as Pearson's χ 2 , with Fisher adjustment or Yate's continuity correction being appropriate. Te univariate and multivariable logistic regression models were used to analyse the change in odds of MetS and HIV status, cART regimen, and clinical and sociodemographic factors. A p value <0.05 was considered statistically signifcant.

General Characteristics of Study Participants.
Te mean age of the study participants was 38.4 ± 13.7 years with twothirds being females. Tere was no diference in mean age among various categories of participants. Tere were high proportions of HIV patients who were hypertensives, underweight, and currently or formerly smoked. Compared to non-HV participants and cART-naïve HIV patients, cARTtreated HIV patients had higher waist circumference, waisthip ratio, percentage body fat, diastolic and mean blood pressure, and heart rate. Te cART-treated HIV patients had higher levels of FPG, triglycerides, and total and LDL cholesterol compared with non-HIV participants (Table 1).

Prevalence of Metabolic Syndrome and Its Components.
Te prevalence of MetS was similar using the NCEP-ATP III and JIS criteria. However, compared to the JIS and NCE-P-ATP III criteria, the prevalence of MetS was higher using the IDF criterion except in cART-treated HIV patients. Irrespective of the criteria used, the prevalence of MetS was higher in cART-treated HIV patients compared to cARTnaïve HIV patients and non-HIV participants. Tere was no diference in the prevalence of MetS between cART-nave HIV patients and non-HIV participants (Figure 1). Subsequently, we chose to use the JIS criterion to defne MetS in the analyses. Concerning the individual components of MetS, IFG, abdominal obesity, low HDL cholesterol, and hypertriglyceridemia were associated with various categories of study participants (Table 2). In logistic regression analyses, compared to non-HIV controls, cART-treated HIV patients had increased odds of having IFG, low HDL cholesterol, hypertriglyceridemia, and MetS in both unadjusted and adjusted models (Table 3).

Factors Associated with Metabolic
Syndrome. Among the entire study participants, age, being cART-treated HIV patient, females, and being self-employed were associated with increased odds of having MetS, whereas having formal education upto senior high school and tertiary was associated with decreased odds of having MetS in unadjusted logistic regression models. In the adjusted logistic regression models, being cART-treated or cART-naïve HIV patients and female gender were associated with increased odds of having MetS (Table 4).

Association between Metabolic Syndrome and cART
Treatment. Te average duration of HIV infection in cARTtreated HIV patients was 7.6 ± 4.6 years, and the average duration of cART treatment was 7.2 ± 4.5 years. For the cART medication regimen, 94 (59.5%) patients were treated with TDF/3TC/NVP or EFV regimens, 52 (32.9%) patients were on AZT/3TC/NVP or EFV regimens, and 12 (7.6%) patients were on LPV/r-based regimens. Patients on the AZT-based regimens had increased odds of having MetS, while those on the TDF-based regimen had decreased odds of having MetS in both unadjusted and adjusted logistic regression models. Patients on the EFV-based regimen had decreased odds of MetS in an unadjusted regression model but not in the adjusted model. Tere was no association between MetS and patients on NVP-based or LVP/r-based regimens (Table 5). Te association between MetS and the components of MetS is shown in Table S1 (Supplementary digital content, online only).

Major Findings.
Te fndings of this study indicate that cART-treated HIV patients had a higher prevalence of MetS compared to cART-naïve HIV patients and non-HIV controls, but no diference in the prevalence of MetS between cART-naïve HIV patients and non-HIV controls. MetS was associated with HIV status and female gender in all study participants. In cART-treated patients, being on the AZT-based regimen was associated with increased odds of having MetS and being on the TDF-based regimen was associated with decreased odds of having MetS.

Prevalence of MetS.
Te prevalence of MetS in our study population is similar to a previous study in Ghana, which reported the prevalence of MetS to be 25.2% in cART-naïve HIV patients and 50.3% in cART-treated HIV patients using the NCEP-ATP III criterion [13]. However, this study did AIDS Research and Treatment      AZT, zidovudine; EVF efavirenz; LPV/r, Lopinavir/ritonavir; NVP, nevirapine; TDF, tenofovir. Lamivudine was part of the medication regimen for 154 (97.5%) patients, and hence, we did not analyse it. Stavudine and emtricitabine were taken by 2 and 4 patients, respectively, and hence, we did not include them in our analyses.  [20]. However, other studies conducted in Sub-Saharan Africa reported a lower prevalence of MetS compared to what was found in our study. For example, in Nigeria, the prevalence of MetS in PLWH was reported to be 12.7%, 17.2%, and 21% by ATP III, IDF, and JIS criteria [21]. Similarly, the reports from studies conducted in Ivory Coast, Burkina Faso, and South Africa have reported the prevalence of MetS in the cART-treated HIV patients to be 6.2%, 18%, and 8.7%, respectively [7,9,10]. Te variations in the prevalence of MetS may be attributed to variations in the criteria used to defne MetS, geocultural diferences in the studied populations, and various levels of exposure to CVDs [11,16]. In addition to HIV infection and cART treatment that were considered to be exposure variables of MetS, other factors such as diet, physical inactivity, stress, urbanization, and epigenetics may contribute to the development and variation in the prevalence of MetS [17]. Unfortunately, we did not measure these factors in this current study.

AIDS Research and
Concerning the components of MetS, impaired fasting glucose, low HDL cholesterol, and hypertriglyceridemia were more common in the cART-treated HIV patients compared to cART-naïve HIV patients or non-HIV controls after adjusting for confounders. Tis observation of glucose and lipid abnormalities is consistent with previous studies among HIV population in Ethiopia [8,22], Zambia [23], and Cameroon [24]. Te HIV accessory protein, viral protein R, and cART combination have been reported to afect insulin sensitivity by regulating hepatic lipid metabolism, leading to hyperglycemia and dyslipidemia [25]. We did not fnd any association between MetS and lifestyle factors such as alcohol intake and cigarette smoking. Tere were conficting reports on the role of alcohol intake on MetS in HIV patients.
In this study, we employed the quantity/frequency approach to measure alcohol exposure within the past year. However, most of the drinkers in our study population, as is the case of most rural and periurban populations in Africa, consumed locally prepared alcoholic beverage without any standard serving volume (example of some response from participants: calabash full and half of medium gourd bottle) and varying ethanol content based on the preparation procedure (e.g., a day/week fermented palm wine and 4-day fermented malt drink). Terefore, we could not quantify the amount of ethanol exposure. We ended up categorizing them as "drinkers" and "nondrinkers." Te "nondrinkers" are lifetime abstainers, who have no recollection of alcohol intoxication, and those classifed as "drinkers" took more than enough alcohol (intoxicated) at least once a month. Tis method of measuring alcohol exposure has masked the role of alcohol consumption and metabolic syndrome in our study. Tere are conficting reports on the relationship between alcohol intake and MetS. Meta-analysis indicates the high prevalence of MetS in people with alcohol use disorders [26], but lower prevalence of MetS was found in those with low and moderate alcohol intake [27,28]. In addition, the timing of drinking alcohol vis-à-vis meal intake may have dramatic efect on the metabolic efect of alcohol [29]. Similar to our fndings, some studies reported no association between smoking and MetS [30,31], whereas others reported an association between these parameters and MetS [7,10]. It should be noted that our study population has few numbers of smokers to draw any reasonable conclusion on the relationship between smoking and MetS.

MetS and cART Regimen.
Considering the cART-treated HIV patients, those on the AZT-based regimens had an increased likelihood of having MetS, while those on the TDFbased regimens had decreased likelihood of having MetS. Tis observation agrees with the fndings of Labhardt et al. who reported that, in South African HIV patients, those on AZTbased regimens had increased likelihood of MetS compared to those on TDF-based regimens [32]. In contrast to our fndings, Sashindran and Singh reported that TDF-based regimens rather increase the likelihood of MetS in Indian HIV patients [5]. Te AZT-based regimen has been shown to cause oxidative damage, a major mechanistic pathway of MetS and CVDs, through the induction of mitochondrial dysfunction by inhibition of DNA polymerase-c activity [33]. Moreover, a metabolomic study showed that the pathway through which the AZT-based regimen leads to oxidative stress is by altering the metabolism of glutamine, glutamate, glutathione, and arginine biosynthesis as well as alanine, aspartate, and glutamate metabolism [11]. Fortunately, it has been demonstrated in a meta-analysis that the TDF-based regimen has better viral suppression and tolerability compared to the AZT-based regimen [34,35].

Limitations of the Study.
Te major limitation of our study is that it was conducted in a single periurban health facility, limiting the generalization of our fndings to the entire Ghanaian population. Furthermore, we collected data cross-sectionally, and hence, we cannot infer causality from our results. We cannot be specifc that HIV infection and/or cART treatment resulted in increased MetS in our study population because it may be possible that some of our study participants had MetS before HIV infection and/or cART treatment. Moreover, we did not measure some infammatory markers which may have explained the possible mechanisms underlining the high levels of MetS observed in the patient groups. We recommend that future studies should utilize a multicentre longitudinal design to investigate the possible mechanisms underlining the high burden of MetS in HIV infection and cART treatment.

Conclusion
In our study population, the prevalence of MetS is high in the cART-treated HIV patients compared to the cART-naïve

Supplementary Materials
Te Table S1 shows the association between the various components of metabolic syndrome and cART regimens from unadjusted and adjusted logistic regression models in cART-treated HIV patients. Te results showed that the TDF-based regimen was associated with decreased odds of having low HDL cholesterol, while the AZT-based regimen was associated with increased odds of having low HDL cholesterol levels in both unadjusted and adjusted models. Tese results are consistent with previous fndings that TDF-based regimens were associated with 20% increase in HDL cholesterol levels in 48 weeks [36] and substituting TDF in cART with tenofovir alafenamide was associated with decrease in HDL cholesterol [37]. In addition, in Malawian HIV patients, it was reported that those with reduced HDL cholesterol in relation to total plasma cholesterol had signifcant increased mortality [38]. (Supplementary Materials)